This invention relates to the fields of molecular biology and oncology. Specifically, the invention provides a molecular marker for use in the diagnosis of pancreatic cancer.
Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Pancreatic adenocarcinoma (PA) belongs to a group of neoplasms which exhibit a relatively high level of incidence and poor prognosis (1). In the United States, PA is the fifth leading cause of cancer-related deaths and has the lowest 5-year survival rate of any cancer (2,3). In the year 2000, for example, an estimated 28,600 deaths will be ascribed to this type of cancer and approximately 28,600 new cases will be diagnosed. The molecular basis underlying the pathogenesis of PA remains unknown. As a result, the disease has an extremely poor prognosis and lacks early diagnostic and therapeutic modalities.
PA has a median survival of 9-12 months and an overall 5-year survival rate of 3% for all stages. At the time of diagnosis, over four-fifths of patients with PA have clinically apparent metastatic disease. Among patients whose disease is considered to be resectable, 80% will die of recurrent tumor within 2 years. Surprisingly, these statistics actually represent a decrease in both the operative mortality and overall morbidity associated with PA. Factors which appear to be improving long-term survival include improved pancreatectomy technique, earlier detection, reduced perioperative mortality and decreased blood transfusions.
Early diagnosis of PA is difficult but essential in order to develop improved treatments and a possible cure for this disease. Currently, the ability to detect early lesions for resection remains a diagnostic challenge despite the advances in diagnostic imaging methods like ultrasonography (US), endoscopic ultrasonography (EUS), dualphase spiral computer tomography (CT), magnetic resonance imaging (MRT), endoscopic retrograde cholangiopancreatography (ERCP) and transcutaneous or EUS-guided fine-needle aspiration (FNA). Furthermore, distinguishing PA from benign pancreatic diseases, especially chronic pancreatitis, is difficult because of the similarities in radiological and imaging features and the lack of specific clinical symptoms for PA.
Over the past decade, a remarkable increase in the knowledge of somatic genetic alterations underlying human pancreatic cancer cells has been recorded. Mutations of the K-ras oncogene (xcx9c90% of PA cases) and the p53 tumor suppressor gene (50-70%) are the most widely studied genetic tumor markers in pancreatic cancer (4,5). In fact, K-ras mutations have been detected in cytological examinations, from cells present in pancreatic juice and stool samples, as well as in the peripheral blood of patients with pancreatic cancer (6-8). The detection of these mutations have also been associated with chronic pancreatitis (9).
Additionally, there are various highly sensitive PCR-based screening tests for detection of pancreatic cancer cells in blood samples. All of these RT-PCR techniques are based on the detection of genes which are predicted to be specific for pancreatic cancer cells in blood samples (10,11). However, the clinical value, specificity and sensitivity of these molecular tumor markers used in the diagnosis of pancreatic adenocarcinoma differ among the various published studies and are still under evaluation.
The most commonly used clinical tumor markers are serum-based immunoassays for blood group-related antigens and glycoprotein markers, such as CA19-9, CA72-4, CA125, and more recently CA242. However, there are contradictory reports about the specificity and sensitivity of these immunoassays. For example, the specificity of the CA19-9 serum assay for detecting pancreatic cancer ranged from 69% to 93%, and the specificity varied between 46% and 98% (12). Unfortunately, CA19-9 antigen also exhibited elevated serum levels in some benign pancreatic diseases (13).
Further studies have determined that serum marker antigens like CA19-9 are oligosaccharide structures present on mucins. Mucins are heavily glycosylated, high molecular weight proteins that are synthesized and expressed by epithelial cells of the gastrointestinal, respiratory and genito-urinary tracts (14-16). The structure of epithelial mucins displays a protein backbone bearing numerous carbohydrate side chains.
To date, 11 different mucins have been described partially or completely (15, 17, 18). These mucins include: MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC11 and MUC12. Alterations in the expression and structure of these mucins have been reported in different cancers of epithelial origin, such as in pancreatic adenocarcinoma tumors and tumor cell lines, where a dysregulation of MUC1 mucin expression has been described (19-21). Additionally, an aberrant expression of MUC4 in pancreatic cancer cells has also been reported (21-23). The pattern of mucin expression was investigated in pancreatic cancer tissues, pancreatic cancer cell lines and tissue samples of chronic pancreatitis in comparison to normal pancreatic tissue specimens. Pancreatic adenocarcinoma was characterized by an aberrant expression of MUC4 mRNA in 70% of the samples whereas chronic pancreatitis and normal pancreatic tissues were MUC4 negative.
There is some evidence that mucins are also expressed in non-epithelial cells. In recent reports, immune cells, especially T-lymphocytes, were shown to express MUC1 (24-26). The function of MUC1 in immune cells is still under investigation, however, it appears that MUC1 can function as a negative regulator of T cell activation (26).
Despite the improvements of the diagnostic techniques and the knowledge about genetic alterations in mucins, the ability to distinguish between pancreatic cancer and chronic pancreatitis is still a clinical problem. A specific diagnostic test for early detection of pancreatic cancer would greatly aid the clinician in the treatment of this disease.
In accordance with the present invention, it has been discovered that peripheral blood mononucleocytes (PBMCs) isolated from pancreatic cancer patients are positive for MUC4 while MUC4 expression is not observed in PBMCs isolated from normal patients or from patients suffering from chronic pancreatitis or other types of cancers. Accordingly, methods are provided for assessing PBMCs for the presence of MUC4 as a new tumor marker for pancreatic cancer.
In one embodiment of the invention, a method for detecting MUC4 encoding nucleic acid in a biological sample is provided. The method entails extracting nucleic acids from a sample, contacting the extracted nucleic acid with oligonucleotide primers which specifically hybridize to MUC4 encoding nucleic acids if any are present, and subjecting the hybridized nucleic acid and primers to conditions suitable for polymerase chain reaction amplification. The reaction product is then assessed for amplified MUC4 nucleic acid. Suitable primers for use in the method have a sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. Suitable methods for characterizing the reaction product include, without limitation, gel electrophoresis, restriction digest mapping, scintillation counting and filter paper assays.
In a further aspect, kits are provided for performing the method described above. Such a kit comprises at least one pair of primers, a polymerase enzyme suitable for use in polymerase chain reaction, buffers and nucleotides suitable for performing amplification reactions, and optionally an instruction protocol.
In another embodiment of the invention, a method is provided for detecting MUC4 protein in a biological sample. This method entails contacting the biological sample with an antibody or fragment thereof having binding affinity for MUC4, such that MUC4-antibody complexes form if MUC4 is present. The MUC4-antibody complex is then isolated. Such complexes may be assessed using methods which include without limitation, flow cytometric analysis, immunochemical detection or localization and immunoblot analysis.
In another aspect of the invention, kits are provided for performing the immunoassay described above. Such a kit comprises an antibody or fragment thereof having binding affinity for MUC4, a detectable label for said antibody and reagents suitable for detecting MUC4-antibody immunocomplexes, if present in the biological sample.
In yet a further aspect of the invention, antibodies immunologically specific for MUC4 are provided. Such antibodies may be monoclonal or polyclonal. Additionally antibody fragments having binding affinity for MUC4 are provided. Such antibody fragments comprise Fab, Fabxe2x80x2, F(abxe2x80x2)2, F(v) and Sfv generated recombinantly. The anti-MUC4 antibodies or fragments thereof may be used to advantage in the immunoassays and kits described above.